Alternative splicing is the primary mechanism for generation of multiple protein isoforms. Regulation of alternative splicing often requires proteins specific to a cell type or developmental stage, but this very diversity has prevented definition of a molecular mechanism of the process. The functional biochemical and biophysical properties of one protein in the splicing of two neuronal pre-mRNAs offers the opportunity to describe a mechanism. Our goal is to understand the mechanism of exon exclusion of the c-src N1 exon and the GABAA receptor ?2 neuron exon by the polypyrimidine tract binding protein (PTB). PTB is known to be necessary and sufficient for exon exclusion of the N1 exon of c-src pre-mRNA in non-neural cells, and is required for exclusion of the neuron exon of the GABAA receptor ?2 pre-mRNA in non-neural cells. PTB binds near the 3'splice sites of these RNA, but how its binding effectively sequesters the splice site is obscure. Equally obscure is the mechanism by which this repression is relieved by other proteins. Specific Aim 1 is devoted to the characterization of PTB complexes formed on the rat GABA pre-mRNA intron/exon. Specific Aim 2 details interactions of PTB with mouse c-src N1 pre-mRNA exon and flanking introns. We use gel mobility shift assays, nitrocellulose filter binding, enzymatic footprinting, fluorescence anisotropy, and NMR to determine binding affinity, stoichiometry, the RNA sites bound by PTB, and the domains of PTB in contact with the RNA, and fluorescence fluctuation spectroscopy to observe the exchange of fluorescently labeled PTB molecules. Our data have led to the testable hypothesis that the two halves of PTB have specialized RNA binding sites. The two N-terminal domains recognize polypyrimidine tracts in structured RNAs, while the two C-terminal domains bind unstructured RNAs;these RNA sites could be on the same or different RNAs. PTB association/dissociation from complexes will be measured in the presence of nPTB, a reported antagonist, to characterize its mechanism of repression relief.